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Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene with numerous partner genes are frequently found in acute myeloid and acute lymphoblastic leukemia.1, 2 Although the pathomechanism of t(4;11)-mediated leukemia is still being discussed, expression of the AF4•MLL fusion was found to enhance the repopulating potential of CD34+ cells and lead to the development of predominantly proB-acute lymphoblastic leukemia in a mouse model.1, 2 The AF4•MLL protein contains cleavage sites for threonine aspartase-1 (Taspase1).1, 2, 3, 4 Upon processing by Taspase1, the AF4•MLL cleavage products form a protein complex resistant to SIAH-mediated degradation and activate oncogenic programs.3, 5 Furthermore, Taspase1 is overexpressed in liquid and solid human cancers, suggesting that Taspase1 is co-opted to promote and sustain tumorigenesis.6 As genetic deletion of Taspase1 in the mouse produced no overt deficiencies,3 inhibition of Taspase1 may offer novel anticancer strategies, including the treatment of leukemias. Human Taspase1 encodes a protease of 420 amino acids cleaving substrates in trans by recognizing a conserved peptide motif (Q3[F,I,L,V]2D1↓G1′x2′D3′D4′).4 Unfortunately, Taspase1's activity is not affected by common protease inhibitors, therefore currently precluding the assessment of its clinical and therapeutic relevance.3, 4, 7
Here, we present our endeavors to target Taspase1's oncogenic potential by (i) overexpressing inactive Taspase1 variants, and (ii) testing a putative Taspase1 inhibitor (Figure 1a).
As the Taspase1 proenzyme is autoproteolytically cleaved and assumed to assemble into an active αββα-heterodimer, we reasoned that overexpressing inactive Taspase1 mutants would inhibit the formation of active protease dimers. To analyze Taspase1's processing of AF4•MLL substrates in living cells, we employed our cell-based biosensor assay4 (Supplementary Figure S1a). Ectopic expression of Taspase1 promoted cleavage and complete nuclear accumulation of the autofluorescent BioTasp protein, containing the AF4•MLL cleavage site. Co-expression of catalytically inactive Taspase1 mutants, in which the catalytic nucleophile, Thr234, was changed into Val (TaspT234V) or Asp233 was mutated into Ala (TaspD233A), resulted in neither cleavage nor nuclear translocation (Figure 1b). Importantly, our assay as well as immunoblot analysis demonstrated that even co-transfecting a ninefold excess of the inactive Taspase1 mutants over the wild-type Taspase1 expression plasmid did not affect Taspase1's processing of the AF4•MLL biosensor (Figures 1b-d; Supplementary Figure S1b). Similar results were obtained using HA-tagged or untagged Taspase constructs, and these results were also confirmed for the Taspase1 targets TFIIA and USF2 (data not shown). Our results demonstrate that enforced expression of inactive Taspase1 mutants, aiming to inhibit formation of active protease dimers, was not inhibitory. One might speculate that Taspase1 is active already as an αβ-monomer, providing a mechanistic explanation why overexpression of inactive mutants was not trans-dominant.
Besides genetic approaches, chemical decoys allowing the targeted inhibition/activation of proteins also allow to dissect and regulate molecular pathomechanisms. Consequently, we next tested (4-[(4-arsonophenyl)methyl]phenyl) arsonic acid (NSC48300), a recently described Taspase1 inhibitor.8 Prior to experimentation, the identity of the used batch of NSC48300 was confirmed by mass spectrometry (Supplementary Figure S2). NSC48300's potential to inhibit Taspase1's processing of the AF4•MLL substrate was examined in adherent and leukemic cell lines. Surprisingly, NSC48300 did not affect Taspase1's trans-cleavage activity, as indicated by the nuclear accumulation of the AF4•MLL biosensor at concentrations ranging from 10 to 500μ (Figures 2a and b; Supplementary Figures S2c and d). The possibility that nuclear accumulation of the biosensor was indirectly mediated through the inhibition of nuclear export by NSC48300 was excluded by microinjection experiments (Supplementary Table S1). Albeit treatment with 500μM NSC48300 impaired cell vitality, this effect was independent of endogenous Taspase1 levels (Supplementary Figures S2e and f). These results were confirmed by immunoblot analysis, revealing that NSC48300 did also not prevent Taspase1's autoprocessing (Figure 2c), and also further confirmed for the Taspase1 targets TFIIA, DPOLZ and USF2 (data not shown). To provide a molecular rationale for the observed lack of inhibition, we performed molecular docking. Albeit high-affinity NSC48300 binding sites in both the active and inactive Taspase1 structure7 were identified, no binding was detectable at or close to the catalytic nucleophile, Thr234 (Figure 2d, data not shown).
Collectively, though NSC48300 interfered with cell migration and invasion,9 was patented as an anti-angiogenic compound, and inhibited the growth of breast and brain tumors in murine models,8 our results show that these effects are not primarily based on the inhibition of Taspase1. The reason why NSC48300 was reported to affect Taspase1 in an in vitro assay8 but not in vivo (this study) remains to be elucidated.
As it will be unlikely to inhibit Taspase1 by using strategies attempting to interfere with its heterodimer formation, experimental and in silico strategies should focus on the identification of specific chemical Taspase1 inhibitors by screening of compound libraries.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Blood Cancer Journal website (http://www.nature.com/bcj)